مطالعه سیتولوژیک Hordeum bulbosum L. در ایران

نویسندگان

گروه زیست‌شناسی، دانشکده علوم، دانشگاه اصفهان، اصفهان، ایران

چکیده

گونه‌ Hordeum bulbosum همواره به عنوان یکی از منابع آللی مفید که می‌تواند در اصلاح غلات زراعی استفاده شود مد نظر است. در این تحقیق، کاریوتیپ 32 نمونه جمعیتی این گونه جمع‌آوری شده از نقاط مختلف ایران مورد بررسی قرار گرفت. تقارن کایوتیپی جمعیت‌ها و ارتباط آن با مناطق جغرافیایی ارزیابی شد. تمامی جمعیت‌های مطالعه شده تتراپلوئید با کاریوتیپ، متقارن بودند و شاخص‌های بررسی شده نشان از اتوتتراپلوئید بودن این گونه دارد. مشاهدات نشان می‌دهد که جمعیت‌های شمال شرقی (گلستان) و شمال غربی (گردنه حیران) نامتقارن‌ترین و جمعیت‌های غربی، متقارن‌ترین کاریوتیپ را دارند. بر اساس این نتایج می‌توان گفت که قدیمی‌ترین جمعیت‌های این گونه در کوه‌های زاگرس و جوان‌ترین آنها در شمال شرق ایران قرار دارند. این گونه احتمالاً از غرب ایران وارد شده و به سمت شرق گسترش یافته است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Cytological study of Hordeum bulbosum L. in Iran

نویسندگان [English]

  • Hamed Khodayari
  • Hojjatolah Saeidi
Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
چکیده [English]

Hordeum bulbosum L. (Poaceae) is considered to be sources of useful alleles which can be used in cereal improvement. Thirty two native Iranian H. bulbosum were collected from different localities and were studied by karyotype analysis. We assessed the karyotype asymmetry of the Iranian bulbous barley populations and analyzed the data to look for their geographic distribution correlations. All of the studied populations were tetraploid (2n=4x=28) and the analysed parameters of karyotype of H. bulbosum support the autopolyploidy origin of the species with nearly symmetric karyotype. The results showed the most asymmetric karyotypes within northeast (Golestan) and northwest (Gardane-e Heiran) populations and the most symmetric karyotyps in populations from the west of Iran. Therefore, it can be assumed that the oldest populations are in the slopes of Zagros Mountains and the youngest germplasms occur in the northeast of this country. It can be concluded that the species originated from the west of Iran and distributed towards east and northeast.

کلیدواژه‌ها [English]

  • Hordeum bulbosum L
  • Iran
  • Karyotype symmetry
  • Tetraploid

 Introduction

The genus Hordeum consists of 32 species (45 taxa in total, including subspecies and cytotypes) including diploid (2n=2x=14), tetraploid (2n=4x=28) and hexaploid (2n=6x=42) cytotypes with a basic chromosome number of x=7 (Bothmer et al., 1995). The genus is classified into five genome groups, namely H, I, X, Y and XI (Taketa et al., 1999). In this study, genome designation followed that of Taketa et al. (2001), namely, H. vulgare and H. bulbosum both carry the H genome, so that H. marinum carries the X genome, while H. murinum has the Y genome, and the 25 remaining species share variants of the I genome (Taketa et al., 2005). H. bulbosum has been recognized as one of the two separate allogamous species ofthe genus, possessing a sporophytic incompatibility system (Bothmer et al., 1995). This species include two well-known cytotypes, diploid and tetraploid, with the latter being more widespread. The tetraploid cytotype is commonly considered as an autopolyploid (HHHH) (Xu and Snape, 1988; Chin, 1941; Papes and Bosiljevac, 1984).

The populations of bulbous barley grow widely in the mountainous and sub mountainous regions of Iran in the north, northeast, northwest, west, southwest and the south (except in the Central Plateau, northern Persian Gulf and southern Caspian Sea shores) (Bor, 1970) with different and under stressful environmental conditions.

Symeonidis et al., (1985) claimed that the chromosome set of bulbous barley originated from Greece which contains 16 metacentric including 4 satellited, 8 sub-metacentric and 4 telo-centric chromosomes. Nasirzadeh and Mirzaie Nadoushan (2005) reported that bulbous barley in north of Fars province is tetraploid with karyotype formulae (6m+1sm).

The aim of the present work was the evaluation of the cytotypes of H. bulbosum in Iran, characterization of the cytological and karyotypic details (numerical parameters) and their correlations with the geographic distribution of H. bulbosum.

 

Materials and Methods

Plant materials

A total of 32 specimens of H. bulbosum were randomly collected from various regions of Iran by the authors and were identified morphologically according to Bothmer et al., (1995) and analysed cytologically (Table 1).

 

Chromosome spread preparation

The seeds were germinated on paper tissue in pertidishes and the root tips selected for cytological experiments. Somatic chromosomes of meristematic root tip cells were treated from germinated seeds based on Agayev (1996) protocol with minor modifications. Briefly, pretreatment was carried out in saturated solution of Monobromonaphthalene, washed in distilled water for 30 min, fixed in Chromic Acid/Formaldehyde mixture (1/1) at about 4 ºC for 24 h, and finally washed under tap water for 3 h. Then the materials were transferred into 70% ethanol solution and kept refrigerated till staining process. For staining, the materials were transferred into distilled water for about 5-6 min and treated with 1N NaOH at 60 ºC for 10 min, washed in distilled water thoroughly for 30 min then stained in aceto-iron hematoxylin at 30 ºC for 24 h, washed in distilled water for at least 30 min, and macerated for 10-15 min in cellulase-pektinase enzyme solution at 37 ºC.

The roots were gently squashed in 45% acetic acid, on a slide glass and were observed and photographed under an Olympus AX-40 light microscope. At least, five cells were screened and the cells with good spread were used for analyzing and constructing karyograms. In order to characterize the karyotypic asymmetry, 5- 10 chromosome spreads from different individuals of each accession were examined. All chromosome sizes were measured with computer–aided program Image Tool 3.0. The parameters measured for each metaphase chromosome spread included Total Chromosome Length of the haploid complement (TCL), Mean Chromosome Length of the haploid complement (MCL), and Total Form percent (TF%: Ratio between the shortest arms of the chromosomes and their total length); the TF% value was considered to be close to 50% in most symmetric karyotypes and less than 50% based on the degree of asymmetry, (Huziwara, 1962), R (Ratio between the longest and the shortest arms of the chromosomes, Siljak-Yakovlev, 1986), S% (equals to length of the shortest chromosome dividede on length of the longest chromosome, Stebbins, 1971), Asymmetry index (AsI%=100 × ΣL/ΣTCL; where l is long arms in chromosome set and TCL is total chromosome length in chromosome set, Arano and Saito, 1980) and Karyotype formulae: according to their arm ratios (long/short) designated by the position of the centromere: 1 (metacentric; M), 1-1.7 (metacentric; m), 1.7-3 (submetacentric; sm), 3-7 (subtelocentric; st), and 7-39 (telocentric; t)] (Levan et al., 1964).

 

Table 1. Accessions of H. bulbosum (HB) collected from different places in Iran.

Accession no.

Region

Altitude (m)

Locality

HB2W

W

690

Ilam, Darehshahr, Shahre bastani

HB3W

W

642

Ilam, Darehshahr, Gharatmalgeh

HB6W

W

1509

Lorestan, Dorud,Siahkoleh

HB14W

W

1931

Lorestan, Khoramabad toward Borujerd, Zagheh

HB22W

W

1703

Ilam, 45 Km Islamabad-e-gharb toward Eivan

HB23W

W

1580

Kermanshah, 40 Km Eivan toward Islamabad-e-gharb

HB24W

W

1292

Ilam, Darehshahr toward Ilam, Mishkhas

HB30SW

SW

2100

Chaharmahal-va-Bakhtyari, Felard, Aboueshagh, Kahriz

HB73SW

SW

1690

Fars, Eghlid to Marvdasht, Dorudzan

HB76SW

SW

1702

Fars, Shiraz, Roknabad

HB77SW

SW

1975

Fars, Shiraz toward Kazeroon, Hoseinieh

HB79SW

SW

2051

Fars, Shiaz toward Kazerun, Dashte Arjan

HB81SW

SW

1050

Fars, Noorabad-e-Mamasany

HB84SW

SW

2050

Kohgiluie-va-Boyerahmad, Babameidan toward Yasooj

HB87SW

SW

1695

Kohgiluie-va- Boyerahmad, 25 KmYasooj toward Isfahan

HB90SW

SW

1752

Chaharmahal-va-Bakhtyari,Broojen toward yasooj, Felard

HB91SW

SW

2240

Chaharmahal va Bakhtyari, Broojen toward Yasooj

HB95N

N

1640

Tehran, Boomehen

HB105NE

NE

1775

Golestan Azadshahr toward Shahrood, Khoshyeilagh

HB106NE

NE

700

Golestan, National Park of Golestan

HB109NE

NE

993

Khorasane Shomali, Bojnourd, Baba aman park

HB202W

W

1193

Ilam, Darehshahr toward Ilam, Pakal-e-Gerab

HB207W

W

1360

Kermanshah, Kermanshah toward Kamyaran, Vermenje

HB208W

W

1741

Kurdistan, Kamyaran toward Sanandaj, Morvarid

HB209W

W

1581

Kurdistan, Sanandaj

HB211W

W

1257

Kurdistan, 15 Km Sarvabad toward Sanandaj

HB212W

W

1222

Kurdistan, Sarvabad

HB213W

W

1249

Kurdestan, around of Zarivar lake

HB215W

W

1587

Kurdistan, Marivan toward Saghez, Sarshio

HB216W

W

1423

Azarbaijane Gharbi, Boukan, Kanitoomar

HB217NW

NW

1822

Azarbaijane Gharbi, Boukan,Mohabad, Gharehbolagh

HB221NW

NW

1537

Gilan, Astara, Heiran

 

Results and Discussion

All of the studied populations were tetraploid (2n=4x=28) and the results of the analyzed parameters of karyotype of H. bulbosum supported the autopolyploidy origin of the species with nearly symmetric karyotype combining four homologous or near homologous genomes that were in accordance with previous reports (Chin, 1941; Morrison, 1959; Xu and Snape, 1988). Karyotype was nearly symmetrical with chromosomes varying in mean total chromosome lengths from 5.22 (in HB90SW from Dasht-e Felard at Chaharmahal va Bakhtiari province) to 15.04 μm (in B3W from Darrehshahr in Ilam province) (Table 2). The descriptions of karyotype formulae and their analyzed parameters results are shown in Tables 2 and 3, respectively.

 

Table 2. Karyotype analysis of the different populations of H. bulbosum species(n=chromosome number, TL=Total haploid chromatin length, MCL=Mean Chromosome Length, SE=Standard Error, TF%=Total Form percent, S%=Length of the shortest chromosome divided on length of the longest chromosome, R=ratio between the longest and the shortest arms of the chromosomes, AsI%=Asymetry index, *=Satellite.

Population

2n

TCL

MCL ± SE

TF%

S%

R

AsI%

Karyotype formulae

HB2W

28

63.68

9.1±1.117

45.1

68.56

1.23

54.89

2M + 5m*

HB3W

28

92.53

13.22±1.56

41.22

70.61

1.45

58.77

6m* + 1sm

HB6W

28

53.79

7.68 ±0.74

41.68

76.57

1.45

58.31

6m* + 1sm

HB14W

28

73.2

10.5±1.341

44.03

70.77

1.33

55.96

1M + 4m* +2sm

HB22W

28

76.84

10.1±1.288

44.15

73.92

1.28

55.84

2M + 5m*

HB23W

28

84.03

12.0 ±1.235

44.34

77.31

1.27

55.658

7m*

HB24W

28

67.6

9.66±0.933

42.42

76.88

1.39

57.573

1M* + 5m + 1sm

HB30SW

28

81.48

11.64±1.01

42.26

79.12

1.397

57.731

6m* + 1sm

HB73SW

28

55.97

7.1±1.261

43.79

59.3

1.297

56.208

1M + 5m* + 1sm

HB76SW

28

76.28

10.9±1.5

40.53

67.12

1.53

59.465

2M* + 4m + 1sm

HB77SW

28

54.38

7.77±1.055

41.28

65.29

1.467

58.716

1M* + 5m + 1sm

HB79SW

28

74.8

10.69±1.344

42.78

67.01

1.397

57.22

1M* + 5m + 1sm

HB81SW

28

59.26

8.47±0.908

39.7

73.52

1.655

60.3

5m* + 1sm + 1st

HB84SW

28

57

8.14±1.234

38.57

63.35

1.616

61.42

5m* + 2sm

HB87SW

28

57.79

8.26±0.94

41.18

71.48

1.56

58.81

5m* + 2sm

HB90SW

28

44.88

6.41±0.7

43.6

71.21

1.345

56.39

2M + 4m + 1sm

HB91SW

28

51.26

7.32±0.85

41.02

72.35

1.541

58.97

1M + 5m* + 1sm

HB95N

28

76.73

10.961±1.278

42.42

74.43

1.395

57.578

6m* + 1sm

HB105NE

28

55.7

7.96±1.04

40.68

68.6

1.491

59.317

6m* + 1sm

HB106NE

28

63.02

9.002±1.41

37.81

64.17

1.687

62.186

4m + 3sm*

HB109NE

28

59.77

8.538±1.147

40.48

68.65

1.543

59.511

6m* + 1sm

HB202W

28

53.08

7.582±0.79

39.44

70.97

1.57

60.55

1M + 3m + 3sm*

HB207W

28

70.06

10.01±1.03

40.1

74.32

1.53

59.006

1M* + 4m + 2sm

HB208W

28

85.41

12.201±1.55

40.86

71.03

1.493

59.138

1M + 5m* + 1sm

HB209W

28

48.86

6.98±0.69

41.17

76.41

1.533

58.821

1M + 4m* + 2sm

HB211W

28

59.86

8.55±1.74

38.79

51.3

1.6

61.209

5m* + 2sm

HB212W

28

53.45

7.64±0.8

42.37

75.57

1.467

57.623

5m* + 2sm

HB213W

28

85.61

12.23 ±1.27

42.42

73.33

1.438

57.575

1M + 5m* +1sm

HB215W

28

67.07

9.58 ±2.1

40.15

48.58

1.565

59.847

1M* + 4m + 2sm

HB216W

28

73.01

10.43 ±1.4

42.5

69.15

1.366

57.498

5m* + 2sm

HB217W

28

66.91

9.56 ±0.85

41.72

81.32

1.432

58.272

1M*+ 5m + 1sm

HB221W

28

62.61

8.94 ±.91

37.93

72.53

1.683

62.066

1M* + 3m + 3sm

 

The morphological characteristics of chromosomes are shown in Figure 1. As presented in Table 2, the metacentric (M and m) chromosomes dominated the observed karyotypes with 79.46% and the second frequency belongs to the submetacentrics (20.09%). Only one population (HB81SW from Noorabad –e Mamasany in Fars province) had a sub-telocentric (st) chromosome with karyotype formulae (5m* + 1sm + 1st). No telocentric chromosome was observed (see Table 2).

   

 

   

 

   

 

Figure 1: Somatic chromosomes (karyotype) of 32 Iranian H. bulbosum (HB) populations (2n=4x=28).Mitosis squash photograph for accessions: HB24W, HB213W and HB221NW with showing Satellitedchromosoms are presented. Scale bar: 20 μm.

 

The populations HB106NE, HB201W and HB221W had 3 sub-metacentric, HB2W, HB22W (2M + 5m*) and HB23W (7m*) without sub-metacentric chromosome and other remaining populations (56.25%) had karyotype formulae of 6m+1sm including six metacentric and one sub-metacentric that were in accordance with Chin (1941), Linde-Laursen et al. (1990), Morrison (1959) and Vahidy and Jahan (1998) (Table 2). Nasirzadeh and Mirzaie Nadoushan (2005) have analyzed the karyotype parameters of H. bulbosum populations and have suggested that they originated from Fars province and showed that their karyotype formulae were 6m+1sm which was partly in agreement with the results of this study. Symeonidis and Lazaros (1985) reported that the karyotype of Greece populations of bulbous barely was 4m+2sm+1t. In this study, we have not found telocentric chromosome in tetraploids indicating that the karyotype of Iranian tetraploid bulbous barley is different from Greece populations. Our results showed that all populations have one metacentric or sub-metacentric satellited chromosome, except for HB90SW (from Dasht-e Felard in Chaharmahal va Bakhtiari province). Two populations (HB106NE from National Park of Golestan and HB202W from Ilam) had one submetacentric satellited chromosome with karyotype formulae (4M+3sm*). The presence of typical SM satellited chromosomes occurred more frequently among the studied populations of the Hordeum bulbosum (Rajhathy et al., 1964; Vosa, 1976; Coucoli and Symeonidis, 1980; Chin, 1941; Linde-Laursen et al., 1990; Morrison, 1959). As noted by Heneen (1977) and the different origin of the materials should be a logical explanation for the observed differences since SAT chromosomes in the Triticeae are well known to evident morphological variation the of shape and the indices among different populations or varieties of one species. The karyotype formulae polymorphism in homologous chromosomes of H. bulbosum could be correlated with their out-breeding nature. No B chromosome was observed among the materials studied.

The highest TL variation was found in HB215W population[SE (standard error) of MCL=2.1μm], and the lowest chromosome length variation was scored in HB209W population(SE of MCL=0.69 μm) (Table 2). The ratio between the longest and the shortest arms (R) ranged from 1.23 HB3W accessionto 1.69 in HB106NE accession(Table 2). Asymmetry Index (AsI%) ranged from 54.89 in HB2W populationto 62.19 in HB106NE population (Table 2). The degree of karyotype asymmetry as indicated by TF% values ranged from 37.1% (HB106NE and HB221NW accessions) to 45.1% (HB2W) (Table 2). As the TF% values were near to 50%, we can conclude that type of chromosomes were metacentric to submetacentric. Also the mean of S% (Stebbins 1971) indicating symmetry index was from 48.58% (HB215W) to 81.32 (HB217W) with mean of 70.1% indicating nearly symmetrical karyotype for H. bulbosum.

Based on the results of this study (the factors studied and the resulted asymmetry indices) HB221NW proved to have the most asymmetric karyotype (with the formulae of 1M* + 3m + 3sm) among the populations studied. Regarding the asymmetry indices observed in HB221NW it could be suggested that the karyotype asymmetry in this population was mainly affected by the place of the centromers rather than length of the chromosomes. HB2W with the least chromosomal arm ratio variability, showed the most symmetric karyotype (with the formulae of 2M + 5m*). Regarding all the analyzed factors, a high similarity were found between HB2W, HB14W, HB22W and HB23W (see Table 2).

The karyotype asymmetry can be a fine appearance of the general morphology of karyotype in plants (Romero Zarco, 1986). As Sharma (1990) has mentioned, symmetrical karyotypes are more primitive than asymmetrical ones and longer chromosomes than shorter ones; median centromers with chromosome arms of equal length are more primitive than chromosomes with arms of unequal length. From the chromosome length point of view, the longest chromosomes were found in HB3W that could be considered as most primitive population. We observed that the most asymmetric karyotypes within northeast populations (e.g. Golestan) and populations of the west of Iran had the most symmetric karyotypes. Therefore considering the above notions and the results of this study, it could be assumed that the oldest populations are in the slopes of Zagros Mountains (west of Iran) and the youngest ones occurred in the northeast of the country (Figure 2).

 

 

 

 

Figure 2: Distribution of collected accessions of Hordeum bulbosum (W=west, SW=southwest, N=north, NE=northeast, NW=northwest). Arrows indicate the distributions direction of H. bulbosum in Iran.

 

This suggestion is in accordance with the conclusion reported by Bothmer, et al. (1995) namely the H. bulbosum (4x) has originated from Greece and then distributed eastwards. Based on these results it can also be concluded that the Western populations (e.g. HB2W, HB14W, HB22W, HB23W and HB90SW) generally possessed the highest chromosomal length and the highest mean TCL (9.87 μm) and the most symmetric karyotypes are the oldest populations and the northeast populations with mean TCL of 8.5 μmare the youngest populations of H. bulbosum in Iran (Figure 2). 

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